Collected (cell pellet mass=9.31 g), sonicated, centrifuged, and purified expression batch 3. Using 1 mL of clarified supernatent per gradient, wash gradients were tested with 2 imidazole concentrations (15mM and 20mM) to account for the protein lost in purification in Week 11 because the wash imidazole concentration is too high or that the stock's wash has an incorrect concentration (see Figure 3 Week 4 for ladder). NaCL and Tris-base concentrations were kept constant and same to the protocol. SDS gel from wash gradient (see Figure 1) indicated 15mM imidazole was ideal to use as insignificant amount of protein was present in that lane (NDM-1 =28kDa).
I then used the approved new imidazole concentration for purification of expression batch 3 from week 11. Gel verified that new imidazole concentration decreased protein loss from wash Figure 2 is the gel (Compare Elution 1 lane to Figure 3 week 11 and Figure 3 Week 4).
Ran FPLC on expression batch 2. Ran FPLC results of batch 2 on Figure 2 SDS gel with Elution 1 and Elution 2 of batch 3.
Figure 1: SDS wash gradient gel with ColorPlus Ladder (see Figure 1 Week 4). Lanes read left to right are Ladder, 15mM imidazole wash, 20mM imidazole wash. No significant band visible at 30 kDa in 15mM imidazole lane.
Figure 2:
SDS gel with ColorPlus Ladder (see Figure 1 Week 4). Lanes read left to right are post-FPLC sample (10uL), Elution 2, Elution 1, ColorPlus Ladder.
Figure 5: FPLC protocol graphical analysis.Tubes 40-47 were taken for reconcentration.
Week11
-Unfortunately GOLD jobs still have not been completed-submitted on 11/4-edit 11/11 canceled jobs and resent
Running low on protein for enzyme assays-Expressed more protein to prepare to run inhibition assays for when compounds come in. Serena's master plate did not seem viable anymore, and our own plate was misplaced. Made 4 new master plates. Expressed in 2 L LB large culture. Incubated large culture for 18 hrs-then added ZnSO4 per Fast protocol to 5 mM final conc.
11/9 lost 1/2 protein due to adding too much base while adjusting pH of supernatent-protein precipitated in tube. Other tube was fine. Proceeded with Ni-NTA purification. Made another batch of small culture on 11/10->11/11 expressed 1 LB of large culture to replace lost protein (expression batch 3).
SDS gel indicated significant protein loss in wash, and Fast protocol refers to adjusting imidazole concentration during purification. A wash gradient (original imidazole conc:30mM) of 15 mM and 20mM was conducted in week 12.
Elution 1:
Figure 1: Elution 1 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column.
Elution 2:
Figure 2 : Elution 2 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column.
Gel indicated contamination in Elution 1 lane.
Figure 3: SDS protein gel run with ColorPlus protein ladder (see week 4 for ladder). Lanes read in the order from left to right: Elution 2, Elution 1, Wash, Flow Through, Ladder. Gel indicated presence of other proteins/contaminants and thus required further purification for Elution 1 (FPLC) Wash also indicated significant loss of protein.
Figure 4: Concentrated Elution 1 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column. Concentrated to 1mL for FPLC.
Week 10
Madeline, Good job keeping up your wikispaces! Keep up the good work. - Suman 11/4/13
Brief Analysis: Future runs will need to consider keep best ligands script under clean up options is 10% of library/#processors, which also equals to 10% slice size. We'll also need to consider making runs earlier in the week to free up queue on Friday. While ordered compounds are mostly GOLD cb306 ranked compounds, further results from HF9 and CB_diversity may impact future research when comparing to the literature's inhibitors (which are few). Other inhibitors worth testing could be inhibitors of class B beta-lactamases but have not been tested as NDM-1 inhibitors. Comparing virtual results with BC and SZ indicated a high degree of accuracy with GOLD in the top ranked ligands.
Table 1: GOLD ranking comparison of top ten ranked ligands for Madeline Jiang, Brendan Chou, and Serena Zadoo using 4EY2 chain A screened with cb306. Duplicate ligands are shaded in pink with red text. MJ and BC autoscale=2. SZ’s protein structure still contained H2O.
Rank
Madeline
Brendan
Serena
1
5531766
5175515
5531766
2
7726180
5531766
7726180
3
7746693
7726180
5175515
4
5467784
7746693
5192853
5
5192853
5467784
5467784
6
5175515
5192853
7676009
7
6565654
7676009
7746693
8
6629394
6433056
6565654
9
6433056
6565654
7842136
10
7842136
7842136
5282931
Table 2:
Compounds ordered for inhibition assays from cb306 top 10 ranked ligands of GOLD (MJ/BC/SZ) and ICM(MJ). Chemical name not found for 5192853. 5490061 and
5101823 were not in the top GOLD ligands but were ranked 3rd and 5th respectively in the ICM dock done by MJ. This table can also be found in Screened Compounds document. Compound ID refers to Chembridge ID.
Table 3.ICM Chembridge Diversity (n=49797) best ranked ligands against NDM-1 PDB structure 4EY2 chain A. Best ligands determined by ICM score threshold =-32. ( n=11 compounds)
Week 9
Analysis:
The activity assays don't indicate a distinct correlation between velocity and concentration such that the Km value can be determined. most of the results indicate sporadic values that are not suggestive of the trend expected. The results should indicate an increase in velocity as Nitrocefin concentration increases. initially,k we thought that the difficulty in determining the Km value came from the linear region of the Absorbance vs Time graph being too short and thus making it difficult to determine an accurate velocity. Thus, we reduced the Nitrocefin concentrations and tried the assay over a larger range of concentrations in our 2nd attempt at the activity assay. We also conducted the experiment with n=3 trials during the experiment in order to show reproducibility. However, the results of the experiment still didn't indicate the data indicative of a curve that could denote km. In order to try to increase the length of the linear region of the Absorbance vs Time curve even further such that it would be easier to determine the velocity per concentration of Nitrocefin, we reduced the enzyme concentration by 1/2, from 2ug to 1ug per 500uL of total reaction mix in the cuvette. The results still indicated poor Km relation and poor velocity vs concentration correlation.
Nice Job with virtual screening data. Try to elaborate on a brief analysis on the nanodrop data. Thank you. -Max 10/07/2013 Great work Madeline. Good captions and analysis of data. Thank you. -Max 10/21/13
Week 8
Reran enzyme assays from last week. Initial absorbance data vs nitrocefin concentrations was good, but initial velocities were not as favorable, as they begin decreasing at 24.75 uM . Since Km = ~25uM, this is not what we expected. Further assays will likely needed to be conducted-possibly manipulating ZnSO4 concentrations? GOLD validation dock of 4EY2 will be conducted this week in order to compare all of the validation docks to each other as well as against PDB structure 3Q6X.
Table 1: Enzyme assay redo data table for NDM-1/nitrocefin. Trials n=3. NDM-1 = 1.07ug/500 uL.
Nitrocefin conc (uM)
Initial Abs
Velocity
10.2
0.105
0.002497
10.2
0.112
0.002049
10.2
0.109
0.002276
15.15
0.153
0.004147
15.15
0.157
0.002177
15.15
0.158
0.003728
20
0.21
0.004277
20
0.221
0.003791
20
0.197
0.003598
24.75
0.29
0.002988
24.75
0.303
0.004193
24.75
0.28
0.003696
30
0.328
0.002859
30
0.353
0.003176
30
0.333
0.003003
34.884
0.406
0.00409
34.884
0.417
0.002988
34.884
0.4
0.002718
40.23
0.462
0.00251
40.23
0.453
0.003384
40.23
0.451
0.002518
45.455
0.519
0.001719
45.455
0.51
0.002433
45.455
0.502
0.002661\
Figure 1 and Figure 2: Graphs of Initial absorbance and Average initial velocity vs Nitrocefin (substrate) concentration shown from Enzyme assay shown. RedTide spec used. Amount of NDM-1 used =1.07 ug/500uL.
Week 7
Continued to run virtual screening. Since now GOLD is accessible, more docks with GOLD were done as well. Enzyme assay conducted on Friday-results indicated our enzyme was active, but the reaction was proceeding too fast to record accurate/reasonable linear values, especially at higher conc of nitrocefin (substrate). Reccomended to increase trials to n=3, reduce [nitrocefin] range and [Ndm-1] range. NDM-1 used =~2ug/uL
Table 1
Validation dock ranking results using GOLD with 10 positives and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain A (with 2 zinc ions in binding site)
Figure 1: Nitrocefin (substrate) spectra confirming unhydrolyzed nitrocefin (wavelength=386 nm). RedTide Spec used and LoggerPro software used.
Figure 2: Initial velocity determination by absorbance/time slope of nitrocefin hydrolysis (enzyme activity).
Nitrocefin (substrate) spectra (wavelength=386 nm) and hydrolyzed nitrocefin spectra (wavelength=485nm). RedTide Spec used and LoggerPro software used.
FIgure 3: Initial velocity vs Concentration of nitrocefin in Enzyme assay (10/11/13).
Week 6
Analysis for Week 5-6: FPLC protocol was conducted successfully-collection of the majority of our protein in tubes 39-48 (see FIgure 2). Used Gold/Hermes to prep NDM-1 PDB structure 3Q6X chain A and ICM to prep 4EY2 chains A and B for validation docking. Future research will involve running enzyme assays from Elution 1 protein and running virtual screening using the cb306 and chembridge diversity libraries after further analysis validation docking images using Pymol.
Table 3:
Validation dock ranking results using ICM with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain B (with 3 zinc ions in binding site)
Table 2: Validation dock ranking results using ICM with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain A (with 2 zinc ions in binding site)
Table 1: Validation dock ranking results using GOLD with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives. PDB stucture=3Q6X
Analysis: The neg2 rankings (other poses not shown) present concerns to the validity of the dock.
Week 5:
Figure 1: Elution 1 nanodrop absorbance readings after Ni-NTA protein purification. Further purification required FPLC protocol (see Week 4 FIgure 1 for evidence of contamination)
Figure 2: FPLC protocol generated graph showing most of protein was concentrated in tubes 39-48.
Figure 3: Absorbance vs wavelength (nm) nanodrop results of Elution 1 post-FPLC. In comparison to Figure 1, the protein concentration is significantly higher.
Weeks 3 & 4 Fall
madeline - good work, the gel looks good. I hope the enzyme is active. Do you have the FPLC graph for this or is it in the next 2 weeks' block? What is up with your grids on the master plates....? Dr. B 100113 -we used premade plates with Kan+suc, so the grids were already there. FPLC graph is for next week. :Madeline Week 4-
Continuing from week 4, we purified and characterized our protein samples. .5 mL of Ni-NTA "slurry" was used after consulting Dr. B. Ran gel with Colorplus protein ladder.
Figure 1- ColorPlus MW ladder shown on left. On right, gel of protein NDM-1 Beta-lactamase after purification. Lanes: Lane 1: ColorPlus Ladder, Land 2: Flow through Lane 3: Wash Lane 4: Elution 1 Lane 5: Elution Lane 6- Soluble Fraction.
Elution 1 lane contained significant amounts of protein of interest (size 28 kDa) or our protein +other proteins of similar MW. Elution 2 only had a small amount of NDM-1 sized proteins. FPLC will needed to be implemented due to presence of other bands in Elution 1 (contamination of other proteins) to have a more purified protein sample.
Week 3-
Using Week's 2 batch of verified NDM-1 plasmid to transform BL21 (DE3) E. Coli as per Transformation & Expression (https://docs.google.com/file/d/0B_Gl3lMyhDsoSWhzZ2FQaXk1dk0/edit) protocol Day 1. Used Kan plates (NDM-1 encodes ampicillin resistance). Made fresh LB and autoclaved for Day 2. SOC media was added before 2 min post-heatshock. Kan plates shown below.
Figure 1: B21(DE3) E.Coli transformation plates with p27bNDM(35a) on LB+Agar+Suc plates+ 50uL of culture. Figure 2: B21(DE3) E.Coli transformation plates with p27bNDM(35a) on LB+Agar+Sucrose plates+ 10uL of culture.
Inoculated 2x100mL LB media in two separate 500 mL flasks, one with 30 ug/mL Kan and another with 50 ug/mL Kan. Had the flasks grow overnight in 37 degrees Celsius. Used leftover autoclaved LB from week 2's protocol to conduct Day 3 of Expression. OD600 of 0.8 was reached instead of .5 due to adding Kan relatively late. Should have used Chipper spec instead of RedTide according to Dr. B because the RedTides are significantly more expensive, and had some trouble calibrating. Used round curvettes. Added the IPTG and ZnSO4 as per Fast protocol ( ZnSO4 and IPTG solutions are added to 0.05 mM and 0.5 mM) and placed in the 25 degree Celsius water bath incubator for ~22 hrs (slightly over Fast protocol) .Glycerol stocks day 1 protocol initiated. Centrifuged down the cells at 4 degrees C and 6300 rpm using the Brown lab's centrifuge using our rotor. Glycerol stocks made by Brendan and placed in cryovials in the -80C fridge (Tube 1: 3.15g Tube 2: 1.98g) Sonicated (room key in lab notebooks drawer with binder clip) for lysis.
Week 1 & 2 for Fall
Madeline - good. Dr. B 090913
9/6
Analysis for Week 1: Plasmid transformation and expression were successful by sequencing and BlastN results (not posted for Week 1). Nanodrop values were reasonable. Future research will then focus on transforming BL21 E. coli with NDM-1 plasmid we made.
Analyzed sequence by blast and comparing to Fast sequence.
5'3' Frame 2 X X X X X Stop X N F X Stop L Stop E G D I H Met K Y L L P T A A A G L L L L A A Q P A Met A Met G Q Q Met E T G D Q R F G D L V F R Q L A P N V W Q H T S Y L D Met P G F G A V A S N G L I V R D G G R V L V V D T A W T D D Q T A Q I L N W I K Q E I N L P V A L A V V T H A H Q D K Met G G Met D A L H A A G I A T Y A N A L S N Q L A P Q E G Met V A A Q H S L T F A A N G W V E P A T A P N F G P L K V F Y P G P G H T S D N I T V G I D G T D I A F G G C L I K D S K A K S L G N L G D A D T E H Y A A S A R A F G A A F P K A S Met I V Met S H S A P D S R A A I T H T A R Met A D K L R A S Q P E L A P E D P E D V E H H H H H H Stop
Figure 1: BlastP results of ORF2 5'3' Frame for BCMJ27 order of NDM-1 plasmid sequencing request. Top sequence from ORF and bottom sequencing from the Fast document
9/4 Submitted Pymol refresher and received DNA sequencing results from core:
T7:
NNNNNNNNNNNNCNTCTAGANTAATTTTNTTTAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGGCCAGCAAATGGAAACTGGCGACCAACGGTTTGGCGATCTGGTTTTCCGCCAGCTCGCACCGAATGTCTGGCAGCACACTTCCTATCTCGACATGCCGGGTTTCGGGGCAGTCGCTTCCAACGGTTTGATCGTCAGGGATGGCGGCCGCGTGCTGGTGGTCGATACCGCCTGGACCGATGACCAGACCGCCCAGATCCTCAACTGGATCAAGCAGGAGATCAACCTGCCGGTCGCGCTGGCGGTGGTGACTCACGCGCATCAGGACAAGATGGGCGGTATGGACGCGCTGCATGCGGCGGGGATTGCGACTTATGCCAATGCGTTGTCGAACCAGCTTGCCCCGCAAGAGGGGATGGTTGCGGCGCAACACAGCCTGACTTTCGCCGCCAATGGCTGGGTCGAACCAGCAACCGCGCCCAACTTTGGCCCGCTCAAGGTATTTTACCCCGGCCCCGGCCACACCAGTGACAATATCACCGTTGGGATCGACGGCACCGACATCGCTTTTGGTGGCTGCCTGATCAAGGACAGCAAGGCCAAGTCGCTCGGCAATCTCGGTGATGCCGACACTGAGCACTACGCCGCGTCAGCGCGCGCGTTTGGTGCGGCGTTCCCCAAGGCCAGCATGATCGTGATGAGCCATTCCGCCCCCGATAGCCGCGCCGCAATCACTCATACGGCCCGCATGGCCGACAAGCTGCGCGCTAGCCAGCCAGAACTCGCCCCGGAAGACCCCGAGGATGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGANTTGGCTGCTGCCACCGCTGANCAATAACTAGCATAACCCCTTGGGGNNCTAAACGGGTCTTGNNGGGTTTTTGCTGAAGGNNNNTATATCCGNATTGGCGAATGGGNNGNNNCNGNAGNNNGCATTNAGNNNGNNNNNNGNNNNCNCNCANCNNNACNCTANNCTTNCNGCNNNANNNCCNNNNNTTCNNTTTCNTNNNNNTNNNNNNCNNNNNTNNNNNNTTTNCNNNANNNNNAANNGGGGNNCNCNNNNNGNNNNNNTNNNNNNNNGNNNNCNNNNNNNNANANNTNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNN
9/2 Followed Midiprep protocol for Tubes A-E and analyzed plasmid concentrations using nanodrop spectroscopy. Sent a sequencing request to the core to confirm DNA sequence of plasmid. Received NDM-1 Fast document from Dr. B.
Nanodrop Results:
Figure 1: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are too low, and the graph indicates a negative 230 value.
Figure 2: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are too high, and the graph indicates a negative
230 value.
Figure 3: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are now as Expected. Used new blanking TE Buffer and
new nanopure water for blanking and calibrating.
Figure 4: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are now as expected. Used new blanking
TE Buffer and new nanopure water for blanking and calibrating.
DNA LIMs sequencing request:
8/31 Came in at 9 am to take out cultures. 40 mLs of culture was pipetted into 50mL conical tubes and centrifuged at 6000xg for 15 min at 4 degrees C. Since protein and plasmid transformation protocols were different, there was ~20 mLs excess in each flask compared to original protocol. Excess was combined into a 5th tube and centrifuged as well. Tubes were then decanted and pellets stored in the -20degree refrigerator. Total time:1.5 hours.
8/30 Made LB in the morning. Autoclaved 1000 mL LB and two flasks at 10:25 am on liquid cycle #5.Cultured 2 separate colonies (1 per plate) into each flask of 100 mL LB and incubated at 1 pm at 37 degrees Celsius. Total time: 2 hours
8/29 Transformed DH5a bacteria to have NDM-1 plasmid gene (p276 with NDM 35) from Fast lab. Plated the bacteria with Brendan and incubated in 37 degrees Celsius. In total 2 plates with Kan. Note: Had difficult time locating certain materials. Time:3 hrs
Week 3
Nanodrop Spec results for ELution 1 YopH. Protein yield=1.16 mg/mL
Week 2
The likely problem was that too much DNA template was pipetted into the pcr tubes as there's a substantial amount of expression that is too large to be the gene.
Week 1
Figure 1: Transformation plate A of E.coli B21(DE23) with 1 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates. Approximately 1648 colonies/ng plasmid. Figure 2: Transformation plate B of E.coli B21(DE23) with 5 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates. Approximately 3072 colonies/ng plasmid.
Figure 3: Transformation plate C of E.coli B21(DE23) with 25 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates. Approximately 689 colonies/25ng .
6.6
DNA Works output file uploaded to Google Docs under title
DNAWorksoutputfile 3Q6X for Klebsiella pneumoniae
Great work Madeline -UM
Week 12
Collected (cell pellet mass=9.31 g), sonicated, centrifuged, and purified expression batch 3. Using 1 mL of clarified supernatent per gradient, wash gradients were tested with 2 imidazole concentrations (15mM and 20mM) to account for the protein lost in purification in Week 11 because the wash imidazole concentration is too high or that the stock's wash has an incorrect concentration (see Figure 3 Week 4 for ladder). NaCL and Tris-base concentrations were kept constant and same to the protocol. SDS gel from wash gradient (see Figure 1) indicated 15mM imidazole was ideal to use as insignificant amount of protein was present in that lane (NDM-1 =28kDa).
I then used the approved new imidazole concentration for purification of expression batch 3 from week 11. Gel verified that new imidazole concentration decreased protein loss from wash Figure 2 is the gel (Compare Elution 1 lane to Figure 3 week 11 and Figure 3 Week 4).
Ran FPLC on expression batch 2. Ran FPLC results of batch 2 on Figure 2 SDS gel with Elution 1 and Elution 2 of batch 3.
Figure 1: SDS wash gradient gel with ColorPlus Ladder (see Figure 1 Week 4). Lanes read left to right are Ladder, 15mM imidazole wash, 20mM imidazole wash. No significant band visible at 30 kDa in 15mM imidazole lane.
Figure 2:
SDS gel with ColorPlus Ladder (see Figure 1 Week 4). Lanes read left to right are post-FPLC sample (10uL), Elution 2, Elution 1, ColorPlus Ladder.
Figure 5: FPLC protocol graphical analysis.Tubes 40-47 were taken for reconcentration.
Week 11
-Unfortunately GOLD jobs still have not been completed-submitted on 11/4-edit 11/11 canceled jobs and resent
Running low on protein for enzyme assays-Expressed more protein to prepare to run inhibition assays for when compounds come in. Serena's master plate did not seem viable anymore, and our own plate was misplaced. Made 4 new master plates. Expressed in 2 L LB large culture. Incubated large culture for 18 hrs-then added ZnSO4 per Fast protocol to 5 mM final conc.
11/9 lost 1/2 protein due to adding too much base while adjusting pH of supernatent-protein precipitated in tube. Other tube was fine. Proceeded with Ni-NTA purification. Made another batch of small culture on 11/10->11/11 expressed 1 LB of large culture to replace lost protein (expression batch 3).
SDS gel indicated significant protein loss in wash, and Fast protocol refers to adjusting imidazole concentration during purification. A wash gradient (original imidazole conc:30mM) of 15 mM and 20mM was conducted in week 12.
Elution 1:
Figure 1: Elution 1 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column.
Elution 2:
Figure 2 : Elution 2 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column.
Gel indicated contamination in Elution 1 lane.
Figure 3: SDS protein gel run with ColorPlus protein ladder (see week 4 for ladder). Lanes read in the order from left to right: Elution 2, Elution 1, Wash, Flow Through, Ladder. Gel indicated presence of other proteins/contaminants and thus required further purification for Elution 1 (FPLC) Wash also indicated significant loss of protein.
Figure 4: Concentrated Elution 1 of NDM-1 expressed on 11/9 in BL21 (DE3) with pET27b+ vector purified with Ni-NTA column. Concentrated to 1mL for FPLC.
Week 10
Madeline, Good job keeping up your wikispaces! Keep up the good work. - Suman 11/4/13
Brief Analysis: Future runs will need to consider keep best ligands script under clean up options is 10% of library/#processors, which also equals to 10% slice size. We'll also need to consider making runs earlier in the week to free up queue on Friday. While ordered compounds are mostly GOLD cb306 ranked compounds, further results from HF9 and CB_diversity may impact future research when comparing to the literature's inhibitors (which are few). Other inhibitors worth testing could be inhibitors of class B beta-lactamases but have not been tested as NDM-1 inhibitors. Comparing virtual results with BC and SZ indicated a high degree of accuracy with GOLD in the top ranked ligands.
Table 1: GOLD ranking comparison of top ten ranked ligands for Madeline Jiang, Brendan Chou, and Serena Zadoo using 4EY2 chain A screened with cb306. Duplicate ligands are shaded in pink with red text. MJ and BC autoscale=2. SZ’s protein structure still contained H2O.
Table 2:
Compounds ordered for inhibition assays from cb306 top 10 ranked ligands of GOLD (MJ/BC/SZ) and ICM(MJ). Chemical name not found for 5192853. 5490061 and
5101823 were not in the top GOLD ligands but were ranked 3rd and 5th respectively in the ICM dock done by MJ. This table can also be found in Screened Compounds document. Compound ID refers to Chembridge ID.
- GOLD Chembridge Diversity dock rerunning
Table 3.ICM Chembridge Diversity (n=49797) best ranked ligands against NDM-1 PDB structure 4EY2 chain A. Best ligands determined by ICM score threshold =-32. ( n=11 compounds)Week 9
Analysis:
The activity assays don't indicate a distinct correlation between velocity and concentration such that the Km value can be determined. most of the results indicate sporadic values that are not suggestive of the trend expected. The results should indicate an increase in velocity as Nitrocefin concentration increases. initially,k we thought that the difficulty in determining the Km value came from the linear region of the Absorbance vs Time graph being too short and thus making it difficult to determine an accurate velocity. Thus, we reduced the Nitrocefin concentrations and tried the assay over a larger range of concentrations in our 2nd attempt at the activity assay. We also conducted the experiment with n=3 trials during the experiment in order to show reproducibility. However, the results of the experiment still didn't indicate the data indicative of a curve that could denote km. In order to try to increase the length of the linear region of the Absorbance vs Time curve even further such that it would be easier to determine the velocity per concentration of Nitrocefin, we reduced the enzyme concentration by 1/2, from 2ug to 1ug per 500uL of total reaction mix in the cuvette. The results still indicated poor Km relation and poor velocity vs concentration correlation.
Nice Job with virtual screening data. Try to elaborate on a brief analysis on the nanodrop data. Thank you. -Max 10/07/2013
Great work Madeline. Good captions and analysis of data. Thank you. -Max 10/21/13
Week 8
Reran enzyme assays from last week. Initial absorbance data vs nitrocefin concentrations was good, but initial velocities were not as favorable, as they begin decreasing at 24.75 uM . Since Km = ~25uM, this is not what we expected. Further assays will likely needed to be conducted-possibly manipulating ZnSO4 concentrations? GOLD validation dock of 4EY2 will be conducted this week in order to compare all of the validation docks to each other as well as against PDB structure 3Q6X.
Table 1: Enzyme assay redo data table for NDM-1/nitrocefin. Trials n=3. NDM-1 = 1.07ug/500 uL.
Figure 1 and Figure 2: Graphs of Initial absorbance and Average initial velocity vs Nitrocefin (substrate) concentration shown from Enzyme assay shown. RedTide spec used. Amount of NDM-1 used =1.07 ug/500uL.
Week 7
Continued to run virtual screening. Since now GOLD is accessible, more docks with GOLD were done as well. Enzyme assay conducted on Friday-results indicated our enzyme was active, but the reaction was proceeding too fast to record accurate/reasonable linear values, especially at higher conc of nitrocefin (substrate). Reccomended to increase trials to n=3, reduce [nitrocefin] range and [Ndm-1] range. NDM-1 used =~2ug/uL
Table 1
Validation dock ranking results using GOLD with 10 positives and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain A (with 2 zinc ions in binding site)
Table 2: Ranking of GOLD virtual screen for validation of 4EY2 chain A (see more details in Table 1)
Table 3: Top 30 ranked ligands with cb306 library using 4EY2 as NDM-1 structure. Program used=ICM.
Figure 1: Nitrocefin (substrate) spectra confirming unhydrolyzed nitrocefin (wavelength=386 nm). RedTide Spec used and LoggerPro software used.
Figure 2: Initial velocity determination by absorbance/time slope of nitrocefin hydrolysis (enzyme activity).
Nitrocefin (substrate) spectra (wavelength=386 nm) and hydrolyzed nitrocefin spectra (wavelength=485nm). RedTide Spec used and LoggerPro software used.
FIgure 3: Initial velocity vs Concentration of nitrocefin in Enzyme assay (10/11/13).
Week 6
Analysis for Week 5-6: FPLC protocol was conducted successfully-collection of the majority of our protein in tubes 39-48 (see FIgure 2). Used Gold/Hermes to prep NDM-1 PDB structure 3Q6X chain A and ICM to prep 4EY2 chains A and B for validation docking. Future research will involve running enzyme assays from Elution 1 protein and running virtual screening using the cb306 and chembridge diversity libraries after further analysis validation docking images using Pymol.
Table 3:
Validation dock ranking results using ICM with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain B (with 3 zinc ions in binding site)
Table 2: Validation dock ranking results using ICM with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives.PDB structure=4EY2 Chain A (with 2 zinc ions in binding site)
Table 1: Validation dock ranking results using GOLD with 10 positives (including original ligand ampicillinoic acid) and 5 negatives (including aspirin). Negative controls from Zinc filtered with Lipinski's rule of 5, same molecular range as original 6 positives. PDB stucture=3Q6X
Analysis: The neg2 rankings (other poses not shown) present concerns to the validity of the dock.
Week 5:
Weeks 3 & 4 Fall
madeline - good work, the gel looks good. I hope the enzyme is active. Do you have the FPLC graph for this or is it in the next 2 weeks' block? What is up with your grids on the master plates....? Dr. B 100113 -we used premade plates with Kan+suc, so the grids were already there. FPLC graph is for next week. :Madeline
Week 4-
Continuing from week 4, we purified and characterized our protein samples. .5 mL of Ni-NTA "slurry" was used after consulting Dr. B. Ran gel with Colorplus protein ladder.
Figure 1-
ColorPlus MW ladder shown on left. On right, gel of protein NDM-1 Beta-lactamase after purification. Lanes: Lane 1: ColorPlus Ladder,
Land 2: Flow through Lane 3: Wash Lane 4: Elution 1 Lane 5: Elution Lane 6- Soluble Fraction.
Elution 1 lane contained significant amounts of protein of interest (size 28 kDa) or our protein +other proteins of similar MW. Elution 2 only had a small amount of NDM-1 sized proteins. FPLC will needed to be implemented due to presence of other bands in Elution 1 (contamination of other proteins) to have a more purified protein sample.
Week 3-
Using Week's 2 batch of verified NDM-1 plasmid to transform BL21 (DE3) E. Coli as per Transformation & Expression (https://docs.google.com/file/d/0B_Gl3lMyhDsoSWhzZ2FQaXk1dk0/edit) protocol Day 1. Used Kan plates (NDM-1 encodes ampicillin resistance). Made fresh LB and autoclaved for Day 2. SOC media was added before 2 min post-heatshock. Kan plates shown below.
Figure 1: B21(DE3) E.Coli transformation plates with p27bNDM(35a) on LB+Agar+Suc
plates+ 50uL of culture.
Figure 2: B21(DE3) E.Coli transformation plates with p27bNDM(35a) on LB+Agar+Sucrose
plates+ 10uL of culture.
Inoculated 2x100mL LB media in two separate 500 mL flasks, one with 30 ug/mL Kan and another with 50 ug/mL Kan. Had the flasks grow overnight in 37 degrees Celsius. Used leftover autoclaved LB from week 2's protocol to conduct Day 3 of Expression. OD600 of 0.8 was reached instead of .5 due to adding Kan relatively late. Should have used Chipper spec instead of RedTide according to Dr. B because the RedTides are significantly more expensive, and had some trouble calibrating. Used round curvettes. Added the IPTG and ZnSO4 as per Fast protocol ( ZnSO4 and IPTG solutions are added to 0.05 mM and 0.5 mM) and placed in the 25 degree Celsius water bath incubator for ~22 hrs (slightly over Fast protocol) .Glycerol stocks day 1 protocol initiated. Centrifuged down the cells at 4 degrees C and 6300 rpm using the Brown lab's centrifuge using our rotor. Glycerol stocks made by Brendan and placed in cryovials in the -80C fridge (Tube 1: 3.15g Tube 2: 1.98g) Sonicated (room key in lab notebooks drawer with binder clip) for lysis.
Week 1 & 2 for Fall
Madeline - good. Dr. B 090913
9/6
Analysis for Week 1: Plasmid transformation and expression were successful by sequencing and BlastN results (not posted for Week 1). Nanodrop values were reasonable. Future research will then focus on transforming BL21 E. coli with NDM-1 plasmid we made.
Analyzed sequence by blast and comparing to Fast sequence.
5'3' Frame 2
X X X X X Stop X N F X Stop L Stop E G D I H Met K Y L L P T A A A G L L L L A A Q P A Met A Met G Q Q Met E T G D Q R F G D L V F R Q L A P N V W Q H T S Y L D Met P G F G A V A S N G L I V R D G G R V L V V D T A W T D D Q T A Q I L N W I K Q E I N L P V A L A V V T H A H Q D K Met G G Met D A L H A A G I A T Y A N A L S N Q L A P Q E G Met V A A Q H S L T F A A N G W V E P A T A P N F G P L K V F Y P G P G H T S D N I T V G I D G T D I A F G G C L I K D S K A K S L G N L G D A D T E H Y A A S A R A F G A A F P K A S Met I V Met S H S A P D S R A A I T H T A R Met A D K L R A S Q P E L A P E D P E D V E H H H H H H Stop
9/4 Submitted Pymol refresher and received DNA sequencing results from core:
T7:
NNNNNNNNNNNNCNTCTAGANTAATTTTNTTTAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGGCCAGCAAATGGAAACTGGCGACCAACGGTTTGGCGATCTGGTTTTCCGCCAGCTCGCACCGAATGTCTGGCAGCACACTTCCTATCTCGACATGCCGGGTTTCGGGGCAGTCGCTTCCAACGGTTTGATCGTCAGGGATGGCGGCCGCGTGCTGGTGGTCGATACCGCCTGGACCGATGACCAGACCGCCCAGATCCTCAACTGGATCAAGCAGGAGATCAACCTGCCGGTCGCGCTGGCGGTGGTGACTCACGCGCATCAGGACAAGATGGGCGGTATGGACGCGCTGCATGCGGCGGGGATTGCGACTTATGCCAATGCGTTGTCGAACCAGCTTGCCCCGCAAGAGGGGATGGTTGCGGCGCAACACAGCCTGACTTTCGCCGCCAATGGCTGGGTCGAACCAGCAACCGCGCCCAACTTTGGCCCGCTCAAGGTATTTTACCCCGGCCCCGGCCACACCAGTGACAATATCACCGTTGGGATCGACGGCACCGACATCGCTTTTGGTGGCTGCCTGATCAAGGACAGCAAGGCCAAGTCGCTCGGCAATCTCGGTGATGCCGACACTGAGCACTACGCCGCGTCAGCGCGCGCGTTTGGTGCGGCGTTCCCCAAGGCCAGCATGATCGTGATGAGCCATTCCGCCCCCGATAGCCGCGCCGCAATCACTCATACGGCCCGCATGGCCGACAAGCTGCGCGCTAGCCAGCCAGAACTCGCCCCGGAAGACCCCGAGGATGTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGANTTGGCTGCTGCCACCGCTGANCAATAACTAGCATAACCCCTTGGGGNNCTAAACGGGTCTTGNNGGGTTTTTGCTGAAGGNNNNTATATCCGNATTGGCGAATGGGNNGNNNCNGNAGNNNGCATTNAGNNNGNNNNNNGNNNNCNCNCANCNNNACNCTANNCTTNCNGCNNNANNNCCNNNNNTTCNNTTTCNTNNNNNTNNNNNNCNNNNNTNNNNNNTTTNCNNNANNNNNAANNGGGGNNCNCNNNNNGNNNNNNTNNNNNNNNGNNNNCNNNNNNNNANANNTNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNN
T7Term:
NNNNNNNNNNNNNNNTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGACATCCTCGGGGTCTTCCGGGGCGAGTTCTGGCTGGCTAGCGCGCAGCTTGTCGGCCATGCGGGCCGTATGAGTGATTGCGGCGCGGCTATCGGGGGCGGAATGGCTCATCACGATCATGCTGGCCTTGGGGAACGCCGCACCAAACGCGCGCGCTGACGCGGCGTAGTGCTCAGTGTCGGCATCACCGAGATTGCCGAGCGACTTGGCCTTGCTGTCCTTGATCAGGCAGCCACCAAAAGCGATGTCGGTGCCGTCGATCCCAACGGTGATATTGTCACTGGTGTGGCCGGGGCCGGGGTAAAATACCTTGAGCGGGCCAAAGTTGGGCGCGGTTGCTGGTTCGACCCAGCCATTGGCGGCGAAAGTCAGGCTGTGTTGCGCCGCAACCATCCCCTCTTGCGGGGCAAGCTGGTTCGACAACGCATTGGCATAAGTCGCAATCCCCGCCGCATGCAGCGCGTCCATACCGCCCATCTTGTCCTGATGCGCGTGAGTCACCACCGCCAGCGCGACCGGCAGGTTGATCTCCTGCTTGATCCAGTTGAGGATCTGGGCGGTCTGGTCATCGGTCCAGGCGGTATCGACCACCAGCACGCGGCCGCCATCCCTGACGATCAAACCGTTGGAAGCGACTGCCCCGAAACCCGGCATGTCGAGATAGGAAGTGTGCTGCCAGACATTCGGTGCGAGCTGGCGGAAAACCAGATCGCCAAACCGTTGGTCGCCAGTTTCCATTTGCTGGCCCATGGCCATCGCCGGCTGGGCAGCGAGGAGCAGCAGANCAGCAGCAGCGGTCGGCAGCAGGTATTTCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGNTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACNCCGNANGCATCGNGGNNGGCATCACCGGNNCCNNNGNTGCNGNTGCTGGCGNCTATATCNNNANNTCNNCNAATGGGGNNNNTNGGGCNCGCCNCTTNGNCTCNTGNANNNNTGTTCNGNNNNNGGGNNNGGNNGNNGNCCCNNNNNNNNNNTNNNNGNNNCNTCNCNTNNNNNNNNNCNNNNNNNCGNNGGNGGNNNNNANNGNNNNCNNCNNNNNGNNNNNNTTNNANNNANNANTNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNGN
9/2 Followed Midiprep protocol for Tubes A-E and analyzed plasmid concentrations using nanodrop spectroscopy. Sent a sequencing request to the core to confirm DNA sequence of plasmid. Received NDM-1 Fast document from Dr. B.
Nanodrop Results:
Figure 1: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are too low, and the graph indicates a negative 230 value.
Figure 2: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are too high, and the graph indicates a negative
230 value.
Figure 3: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are now as Expected. Used new blanking TE Buffer and
new nanopure water for blanking and calibrating.
Figure 4: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are now as expected. Used new blanking
TE Buffer and new nanopure water for blanking and calibrating.
DNA LIMs sequencing request:
8/31 Came in at 9 am to take out cultures. 40 mLs of culture was pipetted into 50mL conical tubes and centrifuged at 6000xg for 15 min at 4 degrees C. Since protein and plasmid transformation protocols were different, there was ~20 mLs excess in each flask compared to original protocol. Excess was combined into a 5th tube and centrifuged as well. Tubes were then decanted and pellets stored in the -20degree refrigerator. Total time:1.5 hours.
8/30 Made LB in the morning. Autoclaved 1000 mL LB and two flasks at 10:25 am on liquid cycle #5.Cultured 2 separate colonies (1 per plate) into each flask of 100 mL LB and incubated at 1 pm at 37 degrees Celsius. Total time: 2 hours
8/29 Transformed DH5a bacteria to have NDM-1 plasmid gene (p276 with NDM 35) from Fast lab. Plated the bacteria with Brendan and incubated in 37 degrees Celsius. In total 2 plates with Kan. Note: Had difficult time locating certain materials. Time:3 hrs
Week 3
Week 2
The likely problem was that too much DNA template was pipetted into the pcr tubes as there's a substantial amount of expression that is too large to be the gene.
Week 1
Figure 1: Transformation plate A of E.coli B21(DE23) with 1 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates.
Approximately 1648 colonies/ng plasmid.
Figure 2: Transformation plate B of E.coli B21(DE23) with 5 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates.
Approximately 3072 colonies/ng plasmid.
Figure 3: Transformation plate C of E.coli B21(DE23) with 25 ng pNIC-Bsa4 +Mptpb plasmid cultured on LB+Amp Agar plates.
Approximately 689 colonies/25ng
.
6.6
DNA Works output file uploaded to Google Docs under title
DNAWorksoutputfile 3Q6X for Klebsiella pneumoniae